JP2004524004A - Modified CEA and uses thereof - Google Patents

Abstract

The present invention relates to immunogenic CEA agonist polypeptides / proteins comprising a modified epitope comprising the amino acid sequence YLSGADLNL, nucleic acids encoding them, vectors and / or cells containing said nucleic acids, and mixtures and / or compositions thereof. Disclose. Also disclosed is a method for inducing a CEA-specific immune response using the substance.

Description

[0001]
Field of the invention
The present invention relates to immunology, in particular novel biologically active modified CEA agonist polypeptides / proteins comprising modified epitopes, nucleic acids encoding them, vectors and / or cells containing said nucleic acids, mixtures of the foregoing and / or Or compositions and their use as immunogens and / or in the treatment of cancer.
Background of the Invention
Throughout the specification, various references have been set forth in parentheses to more fully describe the state of the art to which this invention pertains. The disclosures of those references are incorporated herein by reference.
[0002]
The promise of cancer immunotherapy is based on the identification of tumor-associated antigens that can be recognized by the immune system. In particular, there is great interest in target antigens that induce T cell-mediated reactions. This suggests that cytotoxic T lymphocytes (CTLs) can be expressed in animal models (Kast W. et al (1989) Cell 59: 6035; Greenberg P. (1991) Adv. Immunol. 49: 281) and humans (Boon T.). et al., Annu. Rev. Immunol 12: 337), both of which are based on evidence that they can induce tumor regression.
[0003]
Human carcinoembryonic antigen (CEA) accounts for the majority of colon, rectal, stomach, and pancreatic cancer (Muaro et al. (1985) Cancer Res. 45: 5769), and approximately 50% of breast cancer (Steward et al. 1974) Cancer 33: 1246), and a 180 kD sugar expressed in 70% of lung cancers (Vincent, RG, Chu, TM (1978) J. Thor. Cardiovas. Surg. 66: 320). It is a protein. CEA is also expressed in fetal gastrointestinal tissue and, to a lesser extent, normal colon epithelial tissue. The immunogenicity of CEA is ambiguous, and several studies have reported the presence of anti-CEA antibodies in patients (Gold et al. (1973) Nature New Biology 239: 60; Pompecki, R. (1980) Eur. J. Cancer 16: 973; Ura et al. (1985) Cancer Lett. 25: 283; Fuchs et al. (1988) Cancer Immunol. Immunother. 26: 180), but other studies have reported its existence. (LoGerfo et al. (1972) Int. J Cancer 9: 344; MacSween, JM (1975) Int J. Cancer 15: 246; Chester KA, Begent, HJ. .. 984) Clin Exp Immunol 58: 685). CEA was first reported in 1965 as a cancer-specific fetal antigen in human adenocarcinoma of the gastrointestinal tract (Gold, P., Freeman, 5.0 (1965) Exp. Med. 121; 439). Since then, CEA has been characterized as an overproduced cell surface antigen in almost all solid tumors of the human gastrointestinal tract. The gene for the human CEA protein has been cloned (Oikawa et al (1987) Biochim. Biophys. Res. 142: 511-518; European Patent Application No. 0346710).
[0004]
Despite the vague evidence of CEA immunogenicity as described above, phase I clinical trials with vaccinia-CEA vaccine ("rV-CEA") have shown that CEA-specific cytotoxic T lymphocytes (CTL ) Demonstrated that the response could be induced in humans (Tsang, KY et al. (1995) J. Natl. Cancer Insit 87: 982-990; Tsang KY et al. (1997) Clin. Cancer Res. 3: 2439-2449). As a result of the study, a CEA immunodominant CTL epitope was identified. The 9-mer (ie, YLSGANLNL) was shown to bind to HLA-A2 class I molecules and was named oncofetal antigen peptide-1 ("CAP-1"). Several subsequent studies also demonstrated the ability of the CAP-1 epitope / peptide itself to induce a CEA-specific human CTL response (Alters, SE et al. (1998) J. Immunother. 21: 17-). 26; Tsang, KY et al. (1997) Supra; Zaremba, S. et al. (1997) Cancer Res. 57: 4570-4577). In addition, stable CTL lines derived from cultures of peripheral blood mononuclear cells (PBMC) from patients vaccinated with rV-CEA with CAP-1 and interleukin (IL) -2 have recently been reported (Tsang, KY (1997) Supra).
[0005]
When an immunogenic peptide is modified in a conserved manner (ie, when a hydrophobic amino acid is replaced with a hydrophobic amino acid), the modified peptide is based on molecular shape, charge, and retention of hydrophobicity. Thus, the theory that they tend to have similar immune activities is accepted. More specifically, a study by Madden (Madden et al. (1993) Cell 75: 693) identified specific amino acid selections in peptides for MHC-complex formation, a precursor step of T cell recognition. Madden and other investigators (Rammensee et al., (1995) Immunogenetics 41: 178) claimed that certain amino acid positions in the peptide were available for T cell recognition.
[0006]
Skipper et al. Describe the identification and characterization of natural peptide epitopes of tyrosinase whose peptide sequence differs from that predicted from DNA ((1996) J. Exp. Med. 183: 527). The modified peptide is recognized by tyrosinase-specific human cytotoxic T lymphocytes ("CTL") more efficiently than the direct translation product and has two proteins to be displayed on the cell surface by the HLA-A2.1 molecule. One of the peptides. The modification was the replacement of asparagine by aspartic acid. The authors suggested that N-glycosylation in the endoplasmic reticulum during protein synthesis was followed by post-translational deamination.
[0007]
For the CEA epitope CAP-1, the first and second anchor positions for HLA binding at positions 2, 9, and 1 (of the epitope) are already occupied by preferred amino acids. Thus, Schlom and colleagues have developed CAP-specific CTLs by creating epitope analogs containing single amino acid substitutions for residues that are predicted to interact with the T cell receptor ("TCR") of CTL. An attempt was made to increase the immunogenicity of one. Shows increased immunogenicity over the immunogenicity of the native epitope, but does not show an associated increase in MHC binding itself (ie, acts as an agonist; Zaremba, S. et al (1997) Supra). Such an analog epitope (YLSGADLNL; named CAP-1-6D) was identified.
[0008]
The present invention relates to novel modified CEA agonist polypeptides / proteins comprising modified epitopes, nucleic acids encoding them, vectors containing said nucleic acids, mixtures and / or compositions of the foregoing, and the induction and induction of CEA-specific immune responses. Disclosed are their advantageous uses in treating cancer.
[0009]
Summary of the Invention
The present invention provides novel modified CEA agonist polypeptides / proteins comprising a modified epitope comprising the sequence YLSGADLNL, nucleic acids encoding the same, vectors (eg, recombinant viruses and / or bacteria) and / or cells (eg, recombinant viruses and / or bacteria) containing said nucleic acids. Antigen presenting cells), as well as mixtures and / or compositions of the foregoing. All of the foregoing substances, mixtures, and compositions comprise a CEA protein or fragment thereof; a CEA agonist polypeptide comprising the modified epitope; a normal or modified CEA epitope; or a CEA protein or fragment thereof, a CEA agonist polypeptide, Or a cell that binds or expresses a normal / modified CEA epitope.
[0010]
Thus, in one embodiment of the present invention, a CEA agonist polypeptide / protein is provided comprising a modified CEA epitope, wherein the modified epitope comprises the sequence YLSGADLNL.
[0011]
In another embodiment of the present invention, the CEA agonist polypeptide / protein has the amino acid sequence of SEQ ID NO: 1 (FIG. 1).
[0012]
As mentioned above, embodiments of the present invention include nucleic acids encoding the CEA agonist polypeptides / proteins described above. Accordingly, an embodiment of the present invention includes the nucleic acid sequence of SEQ ID NO: 2 (FIG. 1). In yet another embodiment of the invention, the nucleic acid is a DNA selected from the group consisting of viral nucleic acids, plasmids, bacterial DNA, naked / free DNA, and RNA. In yet another embodiment, the viral nucleic acid is selected from the group consisting of an adenovirus, an alphavirus, and a poxvirus. In yet another embodiment, the poxvirus is selected from the group consisting of an avipoxvirus, a suipoxvirus, and an orthopoxvirus. In yet another embodiment, the poxvirus nucleic acid is selected from the group consisting of TROVAC, NYVAC, ALVAC, MVA, adeno-associated virus (AAV), Wyeth, and PoxvacTC.
[0013]
An additional embodiment of the present invention provides a sequence encoding a CEA agonist polypeptide / protein, in addition to a second sequence encoding at least one substance selected from the group consisting of cytokines, lymphokines, and costimulatory molecules. It is contemplated to include nucleic acids that include.
[0014]
Embodiments of the present invention also provide a vector comprising the nucleic acid of the present invention. In certain embodiments, the vectors are either recombinant viruses or bacteria. In another embodiment, the recombinant virus is selected from the group consisting of an adenovirus, an alphavirus, and a poxvirus. In yet another embodiment, the poxvirus is selected from the group consisting of avipoxvirus, suipoxvirus, and orthopoxvirus; particular embodiments are ALVAC, NYVAC, TROVAC, MVA, Wyeth, and Poxvac-TC. including.
[0015]
The invention further provides a cell expressing the CEA agonist polypeptide / protein of the invention, comprising the nucleic acid of the invention. In yet another embodiment, cells that express a CEA agonist polypeptide / protein also express an MHC HLA class I molecule. In yet another embodiment, the cells expressing the polypeptide are antigen presenting cells.
[0016]
Embodiments of the present invention include mixtures and / or compositions of the CEA agonist polypeptides / proteins, nucleic acids, vectors and cells described above. The mixtures and / or compositions can optionally include an adjuvant.
[0017]
The present invention
(I) CEA protein or a fragment thereof; and / or
(Ii) a CEA agonist polypeptide / protein of the invention; and / or
(Iii) a CEA epitope; and / or
(Iv) a modified CEA epitope; and / or
(V) a cell that expresses a CEA protein or fragment thereof, a CEA agonist polypeptide / protein of the invention, a CEA epitope, a modified CEA epitope; and / or
(Vi) a method of inducing an immune response in an animal against a CEA protein or a fragment thereof, a cell that binds a CEA agonist polypeptide / protein of the present invention, a CEA epitope, a modified CEA epitope; Further provided is a method comprising administering a CEA agonist polypeptide / protein, nucleic acid, vector, cell, or mixture and / or composition of the invention in a sufficient amount.
[0018]
The present invention provides a method of inhibiting a CEA epitope-expressing cancer cell in a patient, comprising administering to the patient a CEA agonist polypeptide / protein, nucleic acid, vector, cell, or mixture and / or composition of the invention. Provide further.
[0019]
In yet another aspect, the invention provides a method of treating cancer, comprising any one of the above methods for inducing an immune response and / or inhibiting a cancer cell that expresses a CEA epitope.
[0020]
Other features and advantages of the invention will be apparent from the following detailed description. However, the detailed description and specific examples that suggest specific embodiments of the present invention are provided for illustrative purposes only, and various changes and modifications that fall within the spirit and scope of the present invention may be described in detail. Should be apparent to those skilled in the art.
[0021]
The invention will be better understood from the description of the drawings.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a CEA agonist polypeptide / protein comprising a modified CEA epitope comprising the sequence YLSGADLNL, a nucleic acid encoding it, a vector and / or a cell comprising said nucleic acid (collectively referred to as the "substances" of the present invention). And mixtures / compositions of the foregoing. All of the aforementioned substances and mixtures / complexes of the present invention bind or express a CEA protein or fragment thereof, a CEA agonist polypeptide / protein of the present invention, a CEA epitope and / or a modified CEA epitope, and / or those described above. Have the ability to induce an immune response against cells that fail. An "immune response" is defined as any reaction of the immune system, for example, either cellular immunity (ie, cytotoxic T lymphocyte mediated) or humoral immunity (ie, antibody mediated). As is known to those skilled in the art, there are various assays / methodologies for assessing and / or monitoring the immune response.
[0023]
In a cellular immune response, tumor-associated antigenic proteins (eg, CEA) are processed into smaller epitope peptides by intracellular proteases, which are subsequently transported to the cell surface, where they are accommodated on the MHC HLA class I molecule (see FIG. 1). Cleft). T cells recognize epitope peptides only if they bind to MHC HLA class I molecules and are displayed on the surface of the appropriate cells. Similarly, in a humoral immune response, proteins will be processed into smaller epitope peptides, which will subsequently bind to MHC HLA class II molecules and be presented on the cell surface (ie, antigen presenting cells). . Such a complex is recognized by appropriate cells of the humoral immune system.
[0024]
As is known to those of skill in the art, short peptides (ie, epitopes) of about 8 to 12 amino acid amino acid sequences from an antigen can bind directly into the HLA class I molecule housing groove without intracellular processing. . As mentioned earlier, many such epitopes from CEA have been identified. Furthermore, those CEA-specific antigenic epitopes have shown the ability to induce an immune response, whereby target cells expressing the appropriate CEA are thawed. The CEA agonist polypeptides / proteins of the present invention (including modified CEA epitopes) have improved immunity compared to the immune response observed when normal / unmodified CEA (including normal epitopes) is used as the immunogen. Induces an immune response (hence the appearance of a CEA "agonist").
[0025]
CEA agonist polypeptides / proteins included in the present invention comprise a modified epitope comprising the amino acid sequence YLSGADLNL (designated "CAP16D"). The corresponding sequence for the native epitope in unmodified CEA is YLSGANLNL (designated "CAP-1"; Zaremba, S. et al. (1997) Cancer Res. 57: 4570). In certain embodiments of the invention, the CEA agonist polypeptide / protein has the amino acid sequence of SEQ ID NO: 1 (FIG. 1). CEA agonist polypeptides / proteins of the present invention are understood to include both precursor and / or mature forms of the polypeptide / protein (see, eg, Oikawa, S. et al. (1987) Biochem. Biophys. Res. Commun. 142: 511-518).
[0026]
CEA agonist polypeptides / proteins of the present invention are prepared using various methods known to those skilled in the art. Thus, such polypeptides can be provided using recombinant DNA techniques known to those of skill in the art. In a known manner, a nucleic acid sequence encoding a CEA agonist polypeptide / protein of the invention can be incorporated into a suitable expression vector (ie, a recombinant expression vector). Potential expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (eg, replication-defective retroviruses, adenoviruses and adeno-associated viruses, lentiviruses, as long as they are compatible with the host cell in which they are used. Herpes virus, pox virus). The expression "the vector is compatible with the host cell" refers to the expression vector comprising a nucleic acid molecule of the invention (described below) and associated regulatory sequences selected based on the host cell used for expression. A sequence is defined as intended to be operably linked to a nucleic acid molecule. "Operably linked" is intended to mean that the nucleic acid is linked to regulatory sequences in a manner that results in expression of the nucleic acid. Suitable regulatory sequences can be from a variety of sources, including bacterial, fungal, or viral genes, and the like (eg, Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA). (1990)). The selection of the appropriate regulatory sequence depends on the host cell chosen and can be readily determined by one skilled in the art. Examples of such regulatory sequences include: transcription promoters and enhancers, RNA polymerase binding sequences, or ribosome binding sequences (including translation initiation signals). Based on the host cell selected and the expression vector used, other additional sequences (eg, an origin of replication, additional DNA restriction sites, enhancers, and sequences that provide for transcription inducibility) can be incorporated into the expression vector. .
[0027]
The above-described expression vector of the present invention may also include a selectable marker gene that facilitates selection of host cells transformed or transfected with the recombinant molecule of the present invention. Examples of selectable marker genes are genes encoding proteins such as, for example, G418 and hygromycin (which confers resistance to certain drugs), β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase.
[0028]
Transcription of the selectable marker gene is monitored by changing the concentration of the selectable marker protein, such as, for example, β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein that confers antibiotic resistance (eg, G418 in neomycin resistance), transformed cells can be selected using an appropriate selection molecule. As is known to those skilled in the art, cells that have incorporated the selectable marker gene will survive, but cells that do not incorporate any such selectable marker will die. This allows to visualize and measure the expression from the recombinant expression vector of the present invention. It will also be appreciated that the selectable marker can be introduced on a separate vector from the nucleic acid of interest.
[0029]
The recombinant expression vector provides for enhanced expression of the polypeptide of the invention; provides for increased solubility of the polypeptide of the invention; and / or acts as a ligand in affinity purification to target recombinant protein. A gene encoding the fusion moiety may be included to aid purification of the fusion. For example, a proteolytic cleavage site can be added to the target recombinant polypeptide to allow purification of the fusion protein followed by separation of the recombinant polypeptide from the fusion moiety.
[0030]
Solid-phase synthesis (Merfield (1964) J. Am. Chem. Assoc. 65: 2149) or synthesis in homogeneous solution (Methods of Organic Chemistry, E. Wansch (Ed.) Vol. 15, pts. I, Vol. CEA agonist polypeptides / proteins of the invention can be prepared by chemical synthesis using techniques well known in protein chemistry, such as Thieme, Stuttgart (1987).
[0031]
An additional embodiment of the present invention includes a nucleic acid encoding a CEA agonist polypeptide / protein as described above. As described herein, "nucleic acids" include (but are not limited to) viral nucleic acids, plasmids, bacterial DNA, naked / free DNA and RNA. Nucleic acids include both single-stranded and double-stranded forms. Accordingly, these nucleic acids include the relevant nucleotide sequence encoding the polypeptide. For the purpose of definition, the “related nucleotide sequence encoding the polypeptide” further includes a complementary nucleic acid sequence.
[0032]
In one embodiment of the invention, the nucleic acid has the sequence shown by SEQ ID NO: 2 (FIG. 1). In another embodiment of the invention, the nucleic acid comprises its sequence (ie, the sequence of SEQ ID NO: 2 (FIG. 1)). Additional embodiments of the invention consist of and / or comprise nucleic acid sequences encoding precursor or mature CEA agonist polypeptides / proteins.
[0033]
Bacterial DNA useful in embodiments of the present invention are known to those of skill in the art. These bacteria include, for example, Shigella, Salmonella, Cholera, Lactobacillus, Calmette-Guerin (BCG), and Streptococcus. In embodiments of the bacterial DNA of the present invention, the nucleic acid of the present invention can be inserted into the bacterial genome, maintained free, or contained on a plasmid.
[0034]
Embodiments of the viral nucleic acids of the present invention can be derived from a poxvirus or other virus, such as, for example, an adenovirus or an alphavirus. Preferably, the viral nucleic acid is not capable of integrating into the recipient animal cells. Elements for expression of the nucleic acid may include a promoter suitable for expression in recipient animal cells.
[0035]
Embodiments of the invention include a poxvirus nucleic acid selected from the group consisting of avipoxvirus, orthopoxvirus, and suipoxvirus nucleic acids. Particular embodiments include poxvirus nucleic acids selected from the group consisting of vaccinia virus nucleic acid, fowlpox virus nucleic acid, canarypox virus nucleic acid, and swinepox virus nucleic acid; particular examples include TROVAC, NYVAC, ALVAC, MVA, And Wyeth, and Poxvac-TC (described in more detail below).
[0036]
The nucleic acids of the invention may further comprise a nucleic acid sequence encoding at least one substance selected from the group consisting of cytokines, lymphokines, and costimulatory molecules. By way of example and not limitation, interleukin 2, interleukin 12, interleukin 6, interferon gamma, tumor necrosis factor alpha, GM-CSF, B7.1, B7.2, ICAM-1, LFA-3, and Cd72.
[0037]
Standard techniques of molecular biology for preparing and purifying nucleic acids that are well known to those of skill in the art can be used in the preparation of embodiments of the present invention (eg, Current Protocols in Molecular Biology, FM Ausubel et al. Eds.), John Wiley and Sons, Inc., NY, USA (1998), Chpts. 1, 2 and 4; Molecular Cloning: A Laboratory Manual (2nd Ed. Ab. Fritsch and T. Maniatis (Eds.), Cold Spring Harbor Laboratory Press, NY, USA (1989), Chpts. See 7).
[0038]
Aspects of the invention further include a vector comprising the nucleic acid described above. In certain embodiments, the vector can be a recombinant virus or bacterium.
[0039]
Adenoviral vectors and methods for their construction have been described (eg, US Pat. Nos. 5,994,132, 5,932,210, and 6,057,158, and WO 98/17783, 97/44475. Nos. 99/61034, 99/50292, 99/27101, 97/20575, 96/40955, and 96/39534, all of which are incorporated herein by reference. )). Alphavirus vectors are also known in the art and can be used in embodiments of the invention (eg, US Pat. Nos. 5,792,462, 5,739,026, 5,843,723, and 5,789,245, and International Publication No. Nos. / 10578, 95/27044, 95/31565, and 98/15636, all of which are incorporated herein by reference. Lentiviruses have also been described (eg, US Pat. Nos. 6,013,516 and 5,994,136, and WO 96/17816, 97/12622, 98/17815, 98/39463, Nos. 98/46083, 99/15641, 99/19501, 99/30742, 99/31251, 98/51810, and 00/000060, all of which are incorporated by reference. Incorporated herein)). Poxvirus vectors that can be used include, for example, avipoxvirus, orthopoxvirus, or suipoxvirus (US Pat. Nos. 5,364,773, 4,603,112, 5,762,938, 5,378,457, 5,494,807, No. 5,550,941; 5,756,103; 5,833,975; and 5,990,091 (all of which are incorporated herein by reference). By homologous recombination known to those skilled in the art, for example, inserting a nucleic acid encoding a CEA agonist polypeptide / protein into the viral genome under conditions suitable for expression in mammalian cells (described below), A poxvirus vector can be obtained that contains a nucleic acid encoding the CEA agonist polypeptide / protein.
[0040]
In one embodiment of the invention, the poxvirus vector is ALVAC (1) or ALVAC (2), both of which are derived from canarypox virus. ALVAC (1) (or ALVAC (2)) has properties that do not replicate productively in non-avian hosts and that its safety profile is considered to be improved. ALVAC (1) is an attenuated canarypox virus-based vector that is a plaque clone derivative of the approved canarypox vaccine, Kanapox (Tartaglia et al. (1992) Virology 188: 217; USA) Patents 5,550,941, 5,756,103, and 5,833,975 (all of which are incorporated herein by reference)). ALVAC (1) has the same general properties as some general properties of Kanapox. ALVAC-based recombinant viruses expressing foreign antigens have also been shown to be effective as vaccine vectors (Tartaglia et al., In AIDS Research Reviews (vol. 3) Koff W., Wong-Stall F. and Kendy. RC (eds.), Marcel Dekker NY, pp. 361-378 (1993a); Tartaglia, J. et al. (1993b) J. Virol. 67: 2370). For example, mice immunized with an ALVAC (1) recombinant expressing the rabies virus glycoprotein are protected against a lethal challenge with rabies virus (Tartaglia, J. et al., (1992) supra), This shows the potential of ALVAC (1) as a vaccine vector. ALVAC-based recombinants include canine distemper virus (Taylor, J. et al. (1992) Virology 187: 321), and rabies virus (Perkus, ME et al., In Combined Vaccines and Standards Medical Certificate). Issues and Perspective, Annals of the New York Academy of Sciences (1994)), and in cats challenged with the feline leukemia virus (Tartaglia 93, al., Al., Al., Al., Al., Al., Al.). And in horses challenged with equine influenza virus (Ta lor, J. et al., In Proceedings of the Third International Symposium on Avian Influenza, Univ. of Wisconsin-Madison, Madison, Wisconsin, pp. 331-335 (1993)), has also been demonstrated to be useful.
[0041]
ALVAC (2) has a second generation ALVAC vector, vaccinia transcription elements E3L and K3L, inserted at the C6 locus (US Pat. No. 5,990,091, incorporated herein by reference). E3L encodes a protein that can specifically bind to dsRNA. The K3L ORF has considerable homology to E1 F-2. In ALVAC (2), the E3L gene is under the transcriptional control of the native promoter, while K3L is under the control of the early / late vaccine H6 promoter. The E3L and K3L genes act to inhibit PKR activity in cells infected with ALVAC (2), resulting in enhanced levels and continuity of foreign gene expression.
[0042]
Another viral vector system involves the use of a native host restricted poxvirus. Chicken bean virus (FPV) is the archetypal virus of the avipoxvirus genus of the Poxviridae family. Avipoxvirus replication is restricted to birds (Matthews, REF (1982) Intervirology, 17: 42-44), and the literature that avipoxviruses cause productive infections in non-birds, including humans. There are no reports. The host restriction provides an inherent safety barrier to transmission of the virus to other species, making the use of avipoxvirus-based vectors attractive for livestock and human applications.
[0043]
FPV has been conveniently used as a vector to express immunogens from poultry pathogens. The virulent avian influenza virus hemagglutinin protein was expressed in FPV recombinants. Following inoculation of chickens and turkeys with the recombinants, a protective immune response against challenge with an allogeneic or heterologous virulent influenza virus was induced (Taylor, J. et (1968) Vaccine 6: 504). A recombinant PFV expressing the surface glycoprotein of Newcastle disease virus has also been developed (Taylor, J. et al. (1990) J. Virol. 64: 1441-1450; Edbauer, C. et al. (1990) Virology. 179: 901; U.S. Patent No. 5,766,599, which is incorporated herein by reference.
[0044]
A highly attenuated vaccinia strain, called MVA, has also been used as a vector for poxvirus-based vaccines. The use of MVA is described in US Pat. No. 5,185,146.
[0045]
Other attenuated poxvirus vectors have been prepared by genetic modification of wild-type strains of the virus. For example, NYVAC vectors have been derived from the Copenhagen strain of vaccinia by deleting certain virulence and host range genes (Tartaglia, J. et al. (1992), supra; US Pat. Nos. 5,364,773 and 5,494,807. (Incorporated by reference herein), has been found to be useful as a recombinant vector in inducing a protective immune response against an expressed foreign antigen.
[0046]
Recombinant viruses can be constructed by methods known in the art (eg, for vaccinia and avipox viruses, US Pat. Nos. 4,769,330; 4,722,848; 4,603,112; 5,110,587; and 5,174,993). No. 5, pp. 139-64, all of which are incorporated herein by reference).
[0047]
In another embodiment of the present invention, live and / or attenuated bacteria can also be used as vectors. For example, a Vibrio cholera mutant strain would be useful as a bacterial vector in an embodiment of the present invention; US Pat. No. 4,882,278 (two ctxA alleles so that a functional cholera toxin is not produced). WO 92/111354 (strains in which the irgA locus has been inactivated by mutation; sudden mutations in a single strain). Mutations can be combined with ctxA mutations), and WO 94/1533 (deletion mutants lacking functional ctxA and attRS1 DNA sequences). The strains can be engineered to express a heterologous antigen, as described in WO 94/19482 (all patents / patent applications mentioned above are incorporated herein by reference). There). WO 92/11361 describes an attenuated Salmonella typhimurium (S. typhimurium) strain engineered for recombinant expression of a heterologous antigen, and its use as an oral immunogen.
[0048]
Other bacterial strains useful as bacterial vectors in embodiments of the present invention include, but are not limited to, Shigella flexinelli, Streptococcus gordonii, and Bacillus Calmette-Guerin (BCG). Nos. 88/6626, 90/05594, 91/131157, 92/1796, and 92/21376, all of which are incorporated herein by reference. ). In embodiments of the bacterial vector of the present invention, the nucleic acid encoding the CEA agonist polypeptide / protein may be inserted into the bacterial genome, remain free, or may be contained on a plasmid. No problem.
[0049]
The present invention further contemplates including vectors comprising nucleic acids encoding at least one substance selected from the group consisting of cytokines, lymphokines, and costimulatory molecules. The nucleic acid can be adjacent to the sequence encoding the CEA agonist polypeptide / protein, or can be encoded on a separate nucleic acid.
[0050]
Cells containing the nucleic acid encoding a CEA agonist polypeptide / protein are also included in another embodiment of the invention. The cells include any potential cells into which the nucleic acid can be introduced and / or transfected (eg, bacteria, COS cells, Vero cells, chicken embryo fibroblasts, tumor cells, and antigen presentation). cell). The choice of method for introduction and / or transfection into cells depends on the unique properties of the nucleic acid (ie, free DNA, plasmid, integrated into the recombinant virus) and will be known to those skilled in the art (eg, , Current Protocols in Molecular Biology, FM Ausubel et al. (Eds.), John Wiley and Sons, Inc., NY, USA, (1998), Copt. Laboratory Manual (2nd Ed), J. Sambrook, EF Fritsch and T. Maniatis (Eds.), Cold Spring Harbor Laboratory Press, NY, .S.A. (1989), are taught in Chpt's. 1,2,3 and 16).
[0051]
The major histocompatibility complex (MHC) class I and class II proteins serve a central immune function in recruiting T-lymphocytes (ie, CD8 + and CD4 + T lymphocytes) of the immune system. MHC class I proteins are expressed in almost all nucleated cell types throughout the human body; MHC class II molecules are mainly expressed in antigen presenting cells (APCs; ie, mononuclear phagocytes, Langerhans-trees). Dendritic cells, and B lymphocytes). These distinct classes of cell surface molecules (ie, class I and class II) present peptides / epitope (resulting from intracellular processing of protein antigens) to T lymphocytes (CD8 + and CD4 + T lymphocytes, respectively), Initiate both cellular and humoral immune responses. Usually, alloantigens, tumor antigens, or virally-derived epitopes / peptides will be presented in the context of MHC class I molecules; extracellular antigens / proteins will be presented in the context of MHC class II molecules. Will. However, in some situations, endogenous antigens may also be presented in the context of MHC class II molecules (such general immunological principles are described, for example, in Encyclopedia of Immunology (2nd Ed.), Peter J. Delves ( Ed.-in-Chief), Academic Press, San Diego, USA, pp. 174-8, 191-8, 1108-13, 1690-709 (1998)).
[0052]
Thus, embodiments of the present invention contemplate cells that have been transfected / transfected with a nucleic acid encoding a CEA agonist polypeptide / protein and express the polypeptide / protein.
[0053]
As further contemplated herein, embodiments of the invention are cells transfected / transfected with a nucleic acid encoding a CEA agonist polypeptide / protein, wherein the cell comprises an MHC HLA molecule (ie, a class I and / or Or cells that also express class II) are contemplated. In another embodiment, the cells are antigen presenting cells, possibly selected from the group consisting of mononuclear phagocytes, Langerhans dendritic cells ("dendritic cells"), and B lymphocytes.
[0054]
Aspects of the invention include CEA agonist polypeptides / proteins, nucleic acids encoding the same, vectors containing the nucleic acids or cells containing the nucleic acids, and cytokines, lymphokines, costimulatory molecules, and nucleic acids encoding them A mixture of at least one substance selected from the group consisting of: Additional embodiments of the present invention include CEA agonist polypeptides / proteins, nucleic acids encoding them, vector cells or mixtures for administration to a subject in a biocompatible form suitable for administration in vivo. It further comprises a pharmaceutical composition. "Biocompatible form suitable for administration in vivo" means a form of the substance to be administered that has a therapeutic effect that outweighs any toxic effect. The administration of a therapeutically active amount of a pharmaceutical composition of the invention, ie, an "effective amount", is defined as an amount that is effective at the dose and for the duration necessary to achieve the desired result of inducing an immune response in a human. You. The therapeutically effective amount of a substance depends on factors such as, for example, the disease state / health, age, sex, and weight of the recipient, and the ability of the particular polypeptide, the nucleic acid encoding it, or the recombinant virus to induce the desired response. Varies by. Adjust the dosage regimen to provide the optimal therapeutic response. For example, several divided doses may be administered daily or periodically and / or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
[0055]
Preparation of a pharmaceutical composition that can be administered to a subject such that an effective amount of the active agent (ie, the CEA agonist polypeptide / protein, nucleic acid encoding it) is mixed in a mixture with a pharmaceutically acceptable vehicle. The compositions described herein can be prepared by methods (known per se). Suitable media are described, for example, in "Handbook of Pharmaceutical Additives" (Michael and Irene Ash, Gower Publishing Limited, Aldershot, England (1995)). On this basis, but not exclusively, the composition comprises a solution of the substance with one or more pharmaceutically acceptable media or diluents, contained in a buffer of appropriate pH and / or It may be isotonic with body fluids. In this regard, reference may be made to US Pat. No. 5,843,456. The compositions can further include an adjuvant (described below).
[0056]
CEA protein or a fragment thereof; and / or
A CEA agonist polypeptide / protein of the invention; and / or
A CEA epitope; and / or
A modified CEA epitope; and / or
A cell that expresses a CEA protein or fragment thereof, a CEA agonist polypeptide / protein of the invention, a CEA epitope, a modified CEA epitope; and / or
A method of inducing an immune response in an animal against a CEA protein or a fragment thereof, a cell that binds a CEA agonist polypeptide / protein of the present invention, a CEA epitope, a modified CEA epitope, wherein the CEA agonist polypeptide / protein or a fragment thereof; The present invention also provides a method comprising administering to the animal a nucleic acid encoding it, a vector or cell containing the nucleic acid, a mixture thereof or a composition of the foregoing (hereinafter collectively referred to as an "immune substance"). Included in the range. As noted above, an "immune response" is defined as any reaction of the immune system, either cellular immunity (ie, cytotoxic T-lymphocytes) or humoral immunity (ie, antibody mediated). Defined. The immune response can be assessed (including but not limited to) antigen-specific cytotoxicity testing, production of cytotoxins, CEA / CEA by in vivo or in vitro assays well known to many skilled in the art. -Remission of tumors expressing the epitope, inhibition of cancer cells expressing the CEA / CEA-epitope, etc.).
[0057]
Another embodiment of the present invention provides a method of inhibiting CEA epitope-expressing cancer cells in a patient, comprising administering to the patient an effective amount of an immunogen of the present invention. As a patient with (but not limited to) a solid cancer that expresses CEA (or an epitope thereof), colon cancer, lung cancer, pancreatic cancer, endometrial cancer, breast cancer, thyroid cancer, melanoma, oral cancer, pharyngeal cancer, seminomas , Hepatocellular carcinoma, cholangiocarcinoma, squamous cell carcinoma, and prostate cancer. Accordingly, aspects / embodiments of the invention per se include methods of treating a patient having a cancer, including those described above for inducing an immune response and / or inhibiting a CEA epitope-expressing cancer cell.
[0058]
As is known to those skilled in the art, animals can be immunized with the immunogens of the present invention via any conventional route. For example, via mucosal (eg, ocular, intranasal, buccal, stomach, lung, intestinal, rectal, vaginal, urinary) surfaces or via parenteral routes (eg, subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal). Mediated immunity. The preferred route depends on the choice of immunogen (ie, polypeptide / nucleic acid, recombinant / virus, composition, etc.). Administration can be effected in a single dose or by repeated doses at intervals. Appropriate doses will be known to those skilled in the art, such as, for example, the immunogen itself (ie, the polypeptides / nucleic acids (and also their particular types), the route of administration, and the condition of the animal to be vaccinated (weight, age, etc.) Depends on the various parameters that are performed.
[0059]
Accordingly, a composition of the invention comprises an effective amount of a CEA agonist polypeptide / protein of the invention, a nucleic acid encoding the same, a vector or cell or recombinant virus containing the nucleic acid, mixtures thereof, and a pharmaceutical composition of the foregoing. A method of inducing an immune response in an animal, comprising administering an agent.
[0060]
As mentioned above, nucleic acids (specifically, plasmids encoding CEA agonist polypeptides / proteins of the invention and / or free / naked DNA and / or RNA) are used to induce an immune response in animals. (E.g., U.S. Patent No. 5,589,466, McDonnell and Askari, NEJM 334: 42-45 (1996); Kowalkzyk and Ertl, Cell Mol. Life Sci. 55: 751-770). Usually, the nucleic acid is in a form that cannot be replicated in the target animal cell and is not integrated into the animal genome. DNA / RNA molecules encoding a CEA agonist polypeptide / protein are usually placed under the control of a promoter suitable for expression in animal cells. Promoters can function ubiquitously or tissue-specifically. Examples of tissue non-specific promoters include the early cytomegalovirus (CMV) promoter (described in US Pat. No. 4,168,062), and the Rous sarcoma virus promoter. The desmin promoter is tissue specific and results in expression in muscle cells. More widely useful vectors have been described (ie, WO 94/21797).
[0061]
For administration of a nucleic acid encoding a CEA agonist polypeptide / protein of the invention, the nucleic acid can encode a precursor or mature form of said polypeptide / protein. When the nucleic acid encodes a precursor, the precursor can be the same species (eg, Oikawa, S. et al. (1987) Supra) or heterologous. In the case of the latter (heterologous), eukaryotic leader sequences such as, for example, the leader sequence of tissue plasminogen activator (tPA) can be used.
[0062]
The nucleic acids of the invention can be formulated for use as immunogens based on various methods known to those of skill in the art. First, naked / free, without any transport medium (eg, anionic liposomes, cationic lipids, microparticles (eg, gold microparticles), precipitants (eg, calcium phosphate), or any other transfection-enhancing agent) May be used. In that case, with or without a carrier, one simply dilutes the nucleic acid with a physiologically acceptable solution (eg, sterile saline or sterile buffered saline). If a carrier is present, the carrier is preferably isotonic, hypotonic, or slightly hypertonic and has relatively low ionic strength (eg, provided by a sucrose solution, such as a solution containing 20% sucrose). .
[0063]
Alternatively, a substance that assists cell uptake and a nucleic acid may be combined. Examples include the following methods: (i) supplementing with a chemical that alters cell permeability (eg, bupivacaine; see, eg, WO 94/16737), (ii) encapsulating in liposomes, or ( iii) Bound with cationic lipids or silica, gold or tungsten microparticles.
[0064]
Cationic lipids are well known in the art and are commonly used for gene transfer. Examples of such lipids include lipofectin (DOTAP (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride) and DOTAP (1,2-bis (oleyloxy) -3- ( Trimethylammonio) propane, DDAB (dimethyldioctadecyl-ammonium bromide), DOGS (dioctadecylamido-loglysylspermine), and for example DC-Chol (3β- (N- (N ′, N′-dimethylaminomethane)-) Cholesterol derivatives such as carbamoyl) cholesterol) are described in EP 187,702, WO 90/11092, US Pat. No. 5,283,185. , WO 91/15501 and 95/26 No. 56, as well as in US Patent No. 5,527, 928. Cationic lipids for gene transfer are preferably neutral lipids such as, for example, DOPE (dioleylphosphatidylethanolamine). (For example, WO 90/11092 pamphlet).
[0065]
Other transfection-enhancing compounds can be added to the cationic liposome-containing formulation. Many of them are described, for example, in WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/2397. For example, spermine derivatives useful for facilitating the transport of DNA across the nuclear membrane (see, eg, WO 93/18759) and membranes such as GALA, gramicidin S, and cationic bile salts -Membrane-permeabilizing compounds (see, for example, WO 93/19768).
[0066]
Gold or tungsten microparticles can also be used for gene delivery (described in WO 91/359 and WO 93/17706). In that case, a needleless injection device (e.g., as described in U.S. Pat. Nos. 4,945,050 and 5,015,580 and WO 94/24263). Microparticle-coated polynucleotides can be injected intradermally or intraepidermally using a "gene gun").
[0067]
Anionic and neutral liposomes are also well known in the art (see, for example, Liposomes: A Practical Approach, RPC New Ed, IRL Press (1990) for a detailed description of how to make liposomes) and include a wide range of polynucleotides, including polynucleotides. Useful for transporting products.
[0068]
Particular embodiments of the above methods (i.e., methods of inducing an immune response and / or inhibiting cancer cells that express a CEA epitope in a patient) include a prime-boost protocol for administering an immunogen of the invention. . More specifically, the protocol includes (but is not limited to) a specific / discrete form of the immunogen (ie, a nucleic acid encoding the immunogen (eg, plasmid, bacterial / viral / free or naked), or the nucleic acid A "priming" step using a vector (i.e., a recombinant virus, a bacterium) comprising the following, followed by an alternative (i.e., different from that used for "priming") form of the immunogen (i. At least one "boost" step comprising administration of a fragment thereof (eg, an epitope peptide), a nucleic acid encoding an immunogen (or a fragment thereof), or a vector comprising said nucleic acid. "Prime-boost" methodology is known to those skilled in the art (e.g., taught in WO 00/00216, 98/58956, 98/56919, and 97/39971). One advantage of the protocol is that it avoids the problem of inducing a neutralizing immune response against the viral vector itself into which the nucleic acid encoding the immunogen or fragment thereof has been inserted (eg, RM Content et al.). (2000) Clin. Cancer Res. 6: 34-41).
[0069]
As is well known to those skilled in the art, regardless of the mode of administration (ie, recombinant virus, nucleic acid, polypeptide), co-immunization with an adjuvant improves the ability of the immunogen to induce an immune response. Adjuvants are described in “Vaccine Design: the Subunit and Adjuvant Approach”, Powell and Newman, eds. , Plenum Press, New York, U.S.A. S. A. Pp. 61-79 and 141-228 (1995). Adjuvants usually increase the immunogenicity of the immunogen, but need not necessarily be immunogenic itself. Adjuvants act to retain the immunogen locally near the site of administration and provide a depot effect that facilitates slow, sustained release of the antigen to cells of the immune system. Adjuvants also attract immune system cells to the reservoir of the immunogen and stimulate those cells to induce an immune response. Accordingly, embodiments of the present invention provide compositions that further include an adjuvant.
[0070]
Desirable properties of an ideal adjuvant include the following properties:
1) No toxicity;
2) be able to stimulate the immune response for a long period;
3) easy to manufacture and stable for long-term storage;
4) If necessary, be able to induce both cellular and humoral immune responses to antigens administered by various routes;
5) have a synergistic effect with other adjuvants;
6) ability to selectively interact with a population of antigen presenting cells (APCs);
7) capable of specifically inducing an appropriate TH1 or TH2 cell-specific immune response;
8) The ability to selectively increase the level of the appropriate antibody isotype (eg, IgA) for the antigen / immunogen.
[0071]
However, many adjuvants are toxic and produce undesirable side effects that make their use inappropriate in humans and many animals. For example, some adjuvants are granulomas, acute and chronic inflammation (Complete Freund's adjuvant, FCA), cell lysis (saponin and pluronic polymers), fever, arthritis, and anterior uveitis (muramyl dipeptide (MDP)) And lipopolysaccharide (LPS)). In fact, only aluminum hydroxide and aluminum phosphate (collectively referred to as alum) are routinely used as adjuvants in human and livestock vaccines. The effectiveness of alum in raising antibody responses to diphtheria and tetanus toxoids is well established. Nevertheless, alum has its limitations. For example, alum is ineffective against influenza vaccines and also induces a cellular immune response with other immunogens inconsistently. Antibodies induced by alum co-antigen are predominantly of the IgG1 isotype in mice, which is not optimal for protection by vaccine substances.
[0072]
Adjuvants are characterized as being "intrinsic" or "extrinsic". Essential adjuvants (eg, lipopolysaccharides) are themselves the whole and normal component of a substance (ie, killed or attenuated bacteria) that is used as a vaccine. Non-essential adjuvants are immunomodulators that are usually formulated non-covalently to an antigen and further formulated to enhance a host immune response.
[0073]
In embodiments of the present invention, the adjuvant can be one or more substances selected from the group consisting of cytokines, lymphokines, and costimulatory molecules. By way of example and not limitation, interleukin 2, interleukin 12, interleukin 6, interferon gamma, tumor necrosis factor alpha, GM-CSF, 87.1, 87.2, ICAM-1, LFA-3, CD72. Is mentioned. Particular examples specifically include GM-CSF as an adjuvant (e.g., U.S. Patent Nos. 5,679,356, 5,904,920, 5,637,483, 5,759,535, and 5,254,534; European Patent Application No. 2111684; And WO 97/28816, all of which are incorporated herein by reference).
[0074]
The use of saponins themselves as adjuvants is also well known (Lacaille-Dubois, M. and Wagner, H (1996) Phytomedicine 2: 363). For example, Quil A (derived from the bark of the South American tree, Quillaja Saponaria Molina), and fractions thereof, have been widely described (ie, US Pat. No. 5,057,540; Kensil, CR (1996)). Crit Rev The Drug Carrier System 12: 1; EP 362279). Hemolytic saponins Q521 and QSI7 (HPLC purified fraction of Quil A) have been described as potent systemic adjuvants (US Pat. No. 5,057,540; EP 362279). The use of QS7 (a non-hemolytic fraction of Quil A), which acts as a potent adjuvant for systemic vaccines, is also described in those references. The use of QS21 has been further described (Kensil et al. (1991) J. Immunol 146: 431). Combinations of Q521 with polysorbate or cyclodextrin are also known (WO 99/10008). Specific adjuvant systems containing fractions of Quil A (eg, QS3I and QS7) are described in WO 96/33739 and WO 96/11711.
[0075]
Another preferred adjuvant / immunostimulant is an immunostimulatory oligonucleotide comprising an unmethylated CpG dinucleotide ("CpG"). CpG is an abbreviation for cytosine-guanosine dinucleotide motif present in DNA. CpG is known in the art as an adjuvant when administered by both systemic and mucosal routes (WO 962555; EP 468520; Davies et al. (1998), J. Am. 160: 87; McCluskie and Davis (1998) J. Immunol 161: 4463). Numerous studies have shown that synthetic oligonucleotides derived from the BCG gene sequence can also induce immunostimulatory effects (both in vitro and in vivo; Krieg, (1995) Nature 374: 546). Detailed analysis of immunostimulatory oligonucleotide sequences has shown that the CG motif must be in a particular sequence, and that such sequences are common in bacterial DNA but rare in vertebrate DNA (eg, Immunostimulatory sequences are often: purines, purines, C, G, pyrimidines, pyrimidines, and the CG motif is unmethylated; however, other unmethylated CpG sequences have also been found to be immunostimulatory, Can also be used in the present invention). As will be apparent to one of skill in the art, the CG motif / sequence can be incorporated into the nucleic acid of the invention itself, or can be on a separate nucleic acid.
[0076]
Various other adjuvants are also taught and are included in embodiments of the present invention. U.S. Pat. No. 4,855,283 to Lockhoff et al., Which is incorporated herein by reference, discloses N-glycosylamides, N-glycosylureas, and N-glycosylamides, each of which is substituted with a sugar residue by an amino acid. Glycolipid analogs such as N-glycosyl carbamates are taught as immunomodulators or adjuvants. Furthermore, Lockhoff et al. Have found that N-linked glycolipid analogs with structural similarity to natural glycolipids, such as phosphoglycolipids and glyceroglycolipids, have strong immunity in both herpes simplex virus and pseudorabies virus vaccines. It was reported that the reaction could be induced (Chem. Int. Ed. Engl. 30: 1611-1620 (1991)).
[0077]
U.S. Pat. No. 4,258,029 to Moloney (incorporated herein by reference) discloses that octadecyltyrosine hydrochloride (OTH) contains tetanus toxoid, and formalin inactivated type I, II, III. It teaches that it functions as an adjuvant when complexed with a poliovirus vaccine. Have reported that octadecyl esters of aromatic amino acids complexed with recombinant hepatitis B surface antigen enhance the host immune response to hepatitis B virus (J. Immunol. 14: 4798). (1990)).
[0078]
The adjuvant compound may be selected from a polymer of acrylic acid or methacrylic acid and a copolymer of maleic anhydride and an alkenyl derivative. Adjuvant compounds are, in particular, polyalkenyl esters of sugars or polyhydric alcohols and cross-linked polymers of acrylic or methacrylic acid. These compounds are known by the term carbomer (Pharmeuropa Vol. 8, No. 2, June 1996). Preferably, the adjuvant solution of the invention, especially the carbomer, is prepared in distilled water, preferably in the presence of saline, and the resulting solution is at an acidic pH. Dilute the stock solution by adding water, preferably saline (9 g / l NaCl) containing the desired amount or a substantial portion of the NaCl (to obtain the desired final concentration), preferably Simultaneous or subsequent neutralization with NaOH. Its solution at physiological pH is used for mixing with vaccines, which are stored especially in lyophilized, liquid or frozen form.
[0079]
Those skilled in the art will appreciate that polyhydroxy having three or more (preferably eight or less) hydroxyl groups, wherein the hydrogen atoms of at least three hydroxyl groups have been replaced by unsaturated aliphatic radicals having two or more carbon atoms. See also U.S. Pat. No. 2,909,462, which describes an acrylic polymer cross-linked with a compound, which is incorporated herein by reference. Preferred radicals are radicals having 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated radical may itself contain other substituents such as, for example, methyl. The product sold under the name Carbopol (BF Goodrich, Ohio, USA) is particularly preferred. The product is cross-linked with allyl sucrose or allyl pentaerythritol. Among them, reference is made to carbopol (eg, 974P, 934P, and 971P). Among copolymers of maleic anhydride and alkenyl derivatives, copolymerized EMA (Monsanto; a copolymer of maleic anhydride and ethylene, which is linearly bonded or cross-linked (for example, cross-linked with divinyl ether) Is preferred). For further explanation of these chemicals, see J.M. Fields et al. Nature, 1960, 186: 778 (incorporated herein by reference).
[0080]
In another aspect of the invention, adjuvants useful for parenteral administration of the immunizing agent include aluminum compounds (eg, aluminum hydroxide, aluminum phosphate, and aluminum hydroxide phosphate); Salts such as calcium, iron or zinc can be used, or the insolubility of acylated tyrosine or acylated sugars, cationic or anionically derivatized polysaccharides, or polyphosphazenes. It can be a suspension. The antigen can be precipitated with the aluminum compound or adsorbed to the aluminum compound based on standard protocols well known to those skilled in the art.
[0081]
Other adjuvants included in embodiments of the present invention include lipid A, especially 3-de-o-acylated monophosphoryl lipid A (3D-MPL), which is manufactured by Ribi Immunochem, Montana. Chemically, the adjuvant is often provided as a mixture of 3-de-o-acylated monophosphoryl lipid A and 4, 5, or 6 acylated chains. The preferred form of 3D-MPL is in the form of a particle formulation having a particle size of less than 0.2 pm in diameter (EP 689 454).
[0082]
Adjuvants for mucosal immunization include bacterial toxins (eg, cholera toxin (CT), Escherichia coli heat-labile toxin (LT), Clostridium difficile toxin A, and pertussis toxin (PT), or a combination, subunit, toxoid, or Mutant). For example, a purified preparation of natural cholera toxin subunit B (CTB) would be useful. Fragments, homologues, derivatives, and fusions of any of these toxins are also suitable as long as they retain adjuvant activity. Variants with reduced toxicity can be used. Mutants have been described (e.g., WO 95/17211 (Arg-7-Lys CT mutant), 96/6272 (Arg-192-Gly LT mutant), and 95/34323 ( Arg-9-Lys and Glu-129-Gly PT variants) specification). Other LT variants include, for example, Ser-63-Lys, Ala-69-Gly, Gly-1 10-Asp, and Glu-1 12-Asp variants. Bacterial monophosphoryl lipid A (MPLA) from other adjuvants, such as bacteria of various origins (eg, Escherichia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexinery), can also be used in mucosal administration of immunologicals.
[0083]
As an adjuvant useful for both mucosal immunity and parenteral immunization, polyphosphazene (for example, WO 95/2415), DC-chol (3β- (N- (N ′, N′-dimethylaminomethane) -carbamoyl) ) Cholesterol (e.g., U.S. Patent No. 5,283,185 and WO 96/14831), and QS-21 (e.g., WO 88/9336).
[0084]
The adjuvant / immunostimulant described therein can be formulated with carriers such as, for example, liposomes, oil-in-water emulsions, and / or metal salts including aluminum salts (eg, aluminum hydroxide). For example, 3D-MPL can be formulated with aluminum hydroxide (as described in EP 689454), or an oil-in-water emulsion (as described in WO 95/17210); Advantageously with cholesterol-containing liposomes (described in WO 96/33739), oil-in-water emulsions (described in WO 95/17210), or alum (described in WO 98/15287). It can be formulated. When formulated in a vaccine, the immunostimulatory oligonucleotide (ie, CpG) is usually covalently linked to the antigen (WO 96/02555; McCluskie and Davis (1998) Supra) along with the free antigen. WO 98/16247), or formulated with carriers such as, for example, aluminum hydroxide or alum (Davies et al Supra; Brazolot-Millan et al (1998) Proc, Natl. Acad. Sci. 95: 15553), administered in free solution.
[0085]
Adjuvant / immunostimulant formulations are also included within the scope of the present invention. For example, blends of monophosphoryl lipid A and saponin derivatives (WO 94/00153, 95/17210, 96/33739, 98/56414, 99/12565, and 99/165). 11214 pamphlet), or more specifically, a blend of QS21 and 3D-MPL (described in WO 94/00153). Combinations of immunostimulatory oligonucleotides and saponins, or combinations of monophosphoryl lipid A (preferably 3D-MPL) and aluminum salts also form strong adjuvants for use in the present invention.
[0086]
The following non-limiting examples illustrate the invention.
[0087]
Example
Example 1: Creation of a modified CEA gene
T-cell epitopes within the native CEA protein include CAP-1; the epitope includes the amino acid sequence YLSGANLNL (Tsang et al. (1995) J. Natl. Cancer Inst 87: 982-990). As mentioned earlier, the immunogenicity of the epitope (in the form of a peptide) is increased by changing the sixth amino acid position of the CAP-1 epitope from N (asparagine) to D (aspartic acid) (Zaremba et al.). al. (1997) Cancer Res. 57: 4570-4577). Mutation of codon AAC (encoding asparagine) to GAC (encoding aspartic acid) using a standard technique for in vivo mutagenesis (Ausubel et al. (1997) Curr. Protocols in Mol. Blot). Introduced a modification to the full-length CEA gene. The resulting modified CEA gene contains the modified epitope YLSGANLNL (the change from N to D is highlighted). The modified CEA gene encoding a protein having aspartic acid (D) in place of asparagine (N) at the sixth amino acid position of the CAP-1 epitope is referred to as CEA (6D) (eg, SEQ ID NO: 1 (FIG. 1) )).
[0088]
Example 2: Generation of recombinant fowlpox, vaccinia, and MVA constructs
Construction of a recombinant poxvirus is achieved by homologous recombination between the poxvirus genomic DNA and a plasmid vector carrying the homologous sequence to be inserted. Plasmid vectors for insertion of foreign sequences into poxviruses are constructed by standard methods of recombinant DNA technology (eg, Ausubel et al. (1997) Curr. Protocols. Mol. Biol). Plasmid vectors contain one or more chimeric genes, each of which contains a poxvirus promoter linked to a protein coding sequence flanked by viral sequences from non-essential regions of the poxvirus genome. Cells infected with the parental poxvirus are transfected with the plasmid, and the chimeric gene on the plasmid is inserted into the viral genome by recombination between the poxvirus sequence on the plasmid and the corresponding DNA in the viral genome. Any of a variety of selections or screens (Mazzara et al. (1993) Meth. Enzymol. 217: 557-581; Jenkins et al. (1991) AIDS Res. Hum. Retroviruses 7: 991-998; Sutter 19 et al. 3.) Select and purify the recombinant virus using Vaccine 12: 1032-1040). Insertion of a foreign gene into the vaccinia genome is usually confirmed by polymerase chain reaction (PCR) analysis. Expression of the foreign gene is demonstrated by Western analysis and / or immunoprecipitation of the expression product.
[0089]
Creation of recombinant poxvirus
To create a recombinant fowlpox virus rF-CEA (6D), a plasmid vector designated pT5071 was constructed to direct the insertion of foreign sequences into the BamHIJ region of the fowlpox genome. The CEA (6D) gene is under the control of the vaccinia 40K promoter (Gritz et al. (1990) J. Virol. 64: 5948-5957). In addition, the E. coli lacZ gene under the control of the fowlpox virus C1 promoter (Jenkins et al. (1991) AIDS Res. Hum. Retroviruses 7: 991-998) is included for screening recombinant offspring. These foreign sequences are flanked by DNA sequences from the BamHI J region of the fowlpox genome. A plaque purified isolate from the fowlpox virus POXVAC-TC strain was used as the parent virus for the recombinant vaccine. Construction of recombinant fowlpox virus was achieved by homologous recombination between the fowlpox sequence in the fowlpox genome and the corresponding sequence in pT5071 in fowlpox virus-infected primary chicken embryo skin cells transfected with pT5071. You. Recombinant virus was identified in situ using a chromogenic assay performed on virus plaques. The assay detects the expression of the lacZ gene product in the presence of halogenated indolyl-β-D-galactoside (Bluo-gal) (Chakrabarti et al. (1985) Mol. Cell Bio 3403-3409). Viral plaques expressing lacZ appear blue against a transparent background. Positive plaques (designated vT233) were taken from the cells of the monolayer and their progeny were plated again. Purification of the desired recombinant was performed by plaque isolation seven times and re-seeding in the presence of Blue-Gal. A schematic diagram of the genomic structure of vT233 is shown in FIG.
[0090]
To generate recombinant vaccinia rV-CEA (6D), a plasmid vector designated pT2146 was constructed to direct the insertion of foreign sequences into the thymidine kinase gene (located in the Hind III J region of the vaccinia genome). The CEA (6D) gene is under the control of the vaccinia 40K promoter and the E. coli lacZ gene is under the control of the fowlpox virus C1 promoter. These foreign sequences are flanked by DNA sequences from the Hind III J region of the vaccinia genome. A plaque purified isolate from the vaccinia virus Wyeth (New York City Board of Health) strain was used as the parental virus for the recombinant vaccine. Construction of a recombinant vaccinia virus is achieved by homologous recombination between the vaccinia sequence in the Wyeth vaccinia genome and the corresponding sequence in pT2146 in vaccinia virus-infected cells transfected with pT2146. Recombinant virus was identified using the chromogenic method for the lacZ gene product (described above). Positive plaques (designated vT237) were taken from the cells of the monolayer and their progeny were plated again. Purification of the desired recombinant was performed by five plaque isolations and reseeding in the presence of Blue-Gal. A schematic diagram of the genomic structure of vT237 is shown in FIG. 2 (B).
[0091]
To generate a recombinant MVA expressing CEA (6D), a plasmid vector was constructed to direct the insertion of foreign sequences into the MVA genome. The CEA (6D) gene and the E. coli lacZ gene were each placed under the control of a poxvirus promoter. These foreign sequences are flanked by DNA sequences from the MVA genome into which the foreign sequences are inserted (eg, deletion III (Sutter et al. (1994) Vaccine 12: 1032-1040)). The generation of recombinant MVA is achieved by homologous recombination between the MVA sequence in the MVA genome and the corresponding sequence in a plasmid vector in MVA-infected cells transfected with plasmid vector. Under selected conditions, recombinant plaques were taken from the monolayer of cells and their progeny were plated again. Further plaque isolation and replating resulted in purification of the desired recombinant virus. A schematic diagram of the genomic structure of the MVA recombinant construct is shown in FIG. 2 (C).
[0092]
Example 3: anAJ VAC (2) -modified CEA-B7 . Preparation of 1 (human) recombinant construct (vCP1586)
To create ALVAC (2) -CEAmod / hB7.1, H6 (Perkins et al. (1989) J. Virol.) In an ALVAC (generic) donor plasmid specific for the C5 insertion site (see below). 63: 3829-3936)-The coding sequence containing the promoted modified CEA and the human 67.1 gene (under the control of a synthetic early / late promoter) were subcloned. Next, using a donor plasmid, the expression cassette was inserted into the C5 insertion locus in the ALVAC (2) genome by in vitro recombination (Piccini et al. (1987) Meth. Enzymol. 153: 545).
[0093]
Modified CEA to ALVAC (2) vector and human B. A locus designated C5 was used for insertion of the 7.1 coding sequence. Due to the C5 locus located within the extensive inverted terminal repeat (ITR) of the viral genome, insertion at that locus results in two copies of the inserted sequence. A schematic diagram of the recombinant construct is shown in FIG. [Preferably, the polypeptide encoded by C5 has no function and the predicted amino acid reading frame encoded in that region does not share significant homology with any protein in the existing protein databases].
[0094]
Details of creation of vCP1586 ALVAC recombinant
(I) Modified CEA:
Sequence (to the Bg / II site) obtained by PCT amplification from plasmid pT2147 (including the full-length sequence of modified CEA (6D)) using the following PCR primers:
HM102: 5'-TTG-TCC-GAG-CTC-TCG-CGA-TAT-CCG-TTA-AGT-TTG-TAT-CGT-AAT-GGA-GTC-TCC-CTC-GGC-CCC-3 '(SEQ ID NO: 3)
HM103: 5'-CCG-GAA-TTC-TCA-CAA-GAT-CTG-ACT-TTA-TGA-C-3 '(SEQ ID NO: 4).
[0095]
The PCR product was digested with SacI and EcoRI to create compatible ends suitable for cloning into pBSK + vector (DH5α cells) digested with SacI, EcoRI, and shrimp-derived alkaline phosphatase. The resulting plasmid (pF102.103) was sequenced to confirm insertion fidelity. The remainder of the CEA (6D) sequence was isolated by digestion of plasmid pT2147 with BglII and SalI. The resulting 1.7 Kbp fragment contained the 3 'end of CEA (6D). The fragment was ligated into pF102.103 plasmid digested with alkaline phosphatase from BglII, SalI, and shrimp, and further introduced into DH5α cells. The resulting plasmid was named p3'H6MCEA.
[0096]
An ALVAC donor plasmid was constructed by transferring the p3'H6-modified CEA sequence fragment from p3'H6MCEA to the ALVAC insertion plasmid NVQH6MC5. NVQH6MC5 was first created by digesting the C5 donor plasmid NVQH6C5LSP-18 with BamHI in its polylinker region. It is then treated with alkaline phosphatase, and the kinased and annealed oligonucleotide SPC5PL1 (5′-GAT-CGT-CGA-CGA-GCT-CGA-ATT-CG-3 ′) (SEQ ID NO: 5) and Ligation to SPC5PL2 (5'-GAT-CCG-AAT-TCG-ACC-TCG-TCG-AC-3 ') (SEQ ID NO: 6), thereby creating plasmid NVQH6MC5.
[0097]
Plasmid p3'H6MCEA was digested with NruI and SalI, the fragment containing the 3'H6 modified CEA sequence was purified, and then ligated into the NVQH6MC5 vector digested with NruI, XhoI and shrimp-derived alkaline phosphatase. The resulting donor plasmid containing the modified CEA coding sequence under the control of the full length H6 promoter was named pAH6MCEA.
[0098]
(Ii) B7.1
The following plasmid was used to fuse the 42K promoter and PCR amplify the complete B7.1 gene from plasmid pT2147:
HMI04: 5'-TTG-TCC-GAG-CTC-GAA-TTC-TTT-ATT-GGG-AAG-AAT-ATG-ATA-ATA-TTT-TGG-GAT-TTC-AAA-ATT-GAA-AAT-ATA -TAA-TTA-CAA-TAT-AAA-ATG-GGC-CAC-ACA-CGG-AGG-CAG-3 '(SEQ ID NO: 7)
HMI05: 5'-ACG-GCA-GTC-GAC-TTA-TAC-AGG-GCG-TAC-ACT-3 '(SEQ ID NO: 8).
[0099]
Primer HMI04 contained the complete sequence of the 42K promoter. The resulting PCR product (designated F104.105) was digested with EcoRI and SalI and ligated into pBSK + vector (SURE cells) digested with EcoRI, SalI, and shrimp-derived alkaline phosphatase. The resulting plasmid was named pF104.105 and sequenced to confirm insertion fidelity.
[0100]
(Iii) Donor plasmid pA2147
A fragment containing the 41K-67.1 coding sequence was excised from pF104.105 by digestion with EcoRI and SalI and subsequently purified. The fragment was ligated into pAH6MCEA plasmid digested with EcoRI, SalI, and shrimp-derived alkaline phosphatase and introduced into SURA cells. The resulting donor plasmid was named pA2147 (see Figure 4 for the sequence of the H6-promote human modified CEA / 42K promote B7.1 cassette of pA2147 for insertion into ALVAC).
[0101]
(Iv) Creation of vCP1586
Recombinant virus vCP1586 was created by recombination between the donor plasmid pA2147 and the ALVAC (2) rescuing virus. The recombinant virus construct contains the vaccinia H6 promote modified human CEA gene sequence and the 42K promoted human 67.1 gene sequence at the C5 locus (see FIGS. 3 and 4 for details of the ALVAC insertion and inserted DNA sequence). . Recombination was performed using methods described in the publications and known to those of skill in the art (Piccini et al. (1987), supra; supra; and / or, for example, US Pat. No. 4,769,330; Other methods described in U.S. Pat. Nos. 4,722,848, 4,603,112, 5,174,993, and 5,110,587, all of which are incorporated herein by reference, are also available.
[0102]
Confirm Insert
(I) Restriction enzyme analysis
Viral genomic DNA was isolated from cells infected with vCP1586 according to methods well known to those skilled in the art (eg, Current Protocols in Molecular Biology, FM Ausubel et al. (Eds.), John Wiley and Sons, Inc.). , NY, USA (1998); Molecular Cloning: A Laboratory Manual (2nd Ed.), J. Sambrook, EF Fritsch and T. Maniatis, Eds., Canada. Laboratory Press, NY, USA (1998)). Viral genomic DNA was digested with restriction endonucleases (HindIII, PstI, BamHI, or Xhol). The obtained DNA fragment was fractionated by electrophoresis using an agarose gel, and visualized by ethidium bromide staining. The insertion of the modified CEA and B7.1 expression cassettes into the C5 locus was confirmed (see, eg, FIG. 3 for a schematic diagram of the Xhol restriction map of vCP1586).
[0103]
(Ii) immunoprecipitation
As previously mentioned, uninfected HeLa cells or ALVAC (2) parent virus (vCP1468), ALVAC-CEA / B7 (vCP307), ALVAC-hB7.1 (vCP11334), ALVAC-CEAmod / hB7.1 (vCP1585). ) Or ALVAC (2) -CEAmod / hB7.1 (vCP1586), and immunoprecipitation analysis was performed using radiolabeled lysates from HeLa cells infected (Taylor et al. (1990) J. Virol. 64: 1441-1450). Briefly, [³⁵Cultured HeLa cells were infected at a multiplicity of infection (moi) of 10 pfu / cell in a methionine and cysteine free culture supplemented with [S] -methionine / cysteine (30 μCi / ml). Cells were lysed 18 hours after infection. Immunoprecipitation was performed using a B7.1-specific monoclonal antibody (HB71; ATCC # 80991). Immunoprecipitates were fractionated on a 10% SDS-polyacrylamide gel. The gel was fixed and treated for 30 minutes with 1 M sodium salicylate for fluorography. The results using the anti-hB7.1 antibody show the expression of human B7 in HeLa cells infected with ALVAC (2) -CEAmod / hB7.1 (vCP1586) and other ALVAC-based recombinants expressing hB7.1. However, no expression was observed in cells infected with the parent virus (FIG. 5).
[0104]
(Iii) Western blot analysis
Any of the parent virus (vCP1468), ALVAC-CEA / B7 (vCP307), ALVAC-CEA (vCP248), ALVAC-CEAmod / hB7.1 (vCP1585), or ALVAC (2) -CEAmod / hB7.1 (vCP1586) Was infected to HeLa cells at a multiplicity of infection (moi) of 10 pfu / cell for 18 hours. Lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes using standard techniques. The blot was incubated with anti-CEA monoclonal antibody Col-1 (dilution 1/1000) followed by HRP-conjugated rabbit anti-mouse antibody and developed using HRP peroxide substrate with DAB / Metal (Pierce). I let it. The results obtained showed the expression of full-length CEA in HeLa cells infected with ALVAC (2) -CEAmod / hB7.1 (vCP1586) and other ALVAC-based recombinants expressing CEA.
[0105]
While the principles of the invention have been illustrated and described in certain and / or preferred embodiments, those skilled in the art should recognize that changes may be made in the arrangement and details of the invention without departing from such principles. is there. We claim all modifications falling within the scope of the following claims. All publications, patents, and patent applications referred to in this publication are provided as if each publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in their entirety. All of which are incorporated herein by reference.
[Brief description of the drawings]
FIG.
FIG. 1 shows the nucleic acid and amino acid sequences of an embodiment of the present invention comprising a modified CEA.
FIG. 2
FIG. 2 shows three schematics containing the genomic structure of certain recombinant fowlpox virus, vaccinia virus, and MVA constructs expressing modified CEA.
FIG. 3
FIG. 3 shows a schematic representation of the XhoI restriction map profile of the ALVAC (2) -CEA (modified) / human B7.1 construct.
FIG. 4
FIG. 4 shows the nucleic acid sequence of the H6-promote CEA (modified) / human B7.1 insertion cassette used to create the construct of FIG.
FIG. 5
FIG. 5 shows the results of immunoprecipitation analysis of HeLa cells infected with various ALVAC recombinant constructs.
FIG. 6
FIG. 6 shows the results of Western blot analysis of HeLa cells infected with various ALVAC recombinant constructs.

Claims (49)

A CEA agonist polypeptide comprising a modified epitope of CEA, wherein the modified epitope comprises the amino acid sequence YLSGADLNL. The CEA agonist polypeptide according to claim 1, which has the amino acid sequence shown in SEQ ID NO: 1. A nucleic acid comprising a nucleic acid sequence encoding a CEA agonist polypeptide according to claim 1 or 2. The nucleic acid according to claim 3, comprising the sequence shown in SEQ ID NO: 2. 4. The nucleic acid according to claim 3, having the sequence shown in SEQ ID NO: 2. The nucleic acid according to any one of claims 3 to 5, wherein the nucleic acid is selected from the group consisting of viral nucleic acid, bacterial DNA, plasmid DNA, naked / free DNA, and RNA. The nucleic acid of claim 6, wherein the nucleic acid is selected from the group consisting of adenovirus nucleic acid, alphavirus nucleic acid, and poxvirus nucleic acid. The nucleic acid of claim 7, wherein the nucleic acid is selected from the group consisting of avipoxvirus nucleic acid, orthopoxvirus nucleic acid, and suipoxvirus nucleic acid. The nucleic acid according to claim 7, wherein the nucleic acid is selected from the group consisting of vaccinia virus nucleic acid, fowlpox virus nucleic acid, canarypox virus nucleic acid, and swinepox virus nucleic acid. The nucleic acid according to claim 7, wherein the nucleic acid is selected from the group consisting of TROVAC, NYVAC, ALVAC, MVA, Wyeth, and Poxvac-TC. The nucleic acid according to any one of claims 3 to 10, further comprising a nucleic acid sequence encoding at least one substance selected from the group consisting of a cytokine, a lymphokine, and a costimulatory molecule. A vector comprising the nucleic acid according to any one of claims 3 to 11. 13. The vector according to claim 12, wherein the vector is selected from the group consisting of a recombinant virus and a bacterium. 14. The vector according to claim 13, wherein the vector is selected from the group consisting of adenovirus, alphavirus, and poxvirus. The vector according to claim 14, wherein the vector is selected from the group consisting of avipoxvirus, orthopoxvirus, and suipoxvirus. 15. The vector according to claim 14, wherein the vector is selected from the group consisting of vaccinia virus, fowlpox virus, canarypox virus, and swinepox virus. 15. The vector according to claim 14, wherein the vector is selected from the group consisting of TROVAC, NYVAC, ALVAC, MVA, Wyeth, and Poxvac-TC. 18. The vector according to any one of claims 12 to 17, further comprising a nucleic acid sequence encoding at least one substance selected from the group consisting of cytokines, lymphokines, and costimulatory molecules. A cell expressing a CEA agonist polypeptide, comprising a nucleic acid according to any one of claims 3 to 11. The cell according to claim 19, which further expresses an MHC HLA class I molecule. 20. The cell according to claim 19, which is an antigen presenting cell. 22. The cell according to claim 21, which is a dendritic cell. A mixture comprising a nucleic acid according to any one of claims 3 to 11 and at least one substance selected from the group consisting of cytokines, lymphokines, costimulatory molecules and nucleic acids encoding them. A mixture comprising a vector according to any of claims 12 to 18 and at least one substance selected from the group consisting of cytokines, lymphokines, costimulatory molecules and nucleic acids encoding them. A mixture comprising the CEA agonist polypeptide of claim 1 or 2 and at least one substance selected from the group consisting of cytokines, lymphokines, costimulatory molecules, and nucleic acids encoding them. 23. A mixture comprising cells according to any one of claims 19 to 22 and at least one substance selected from the group consisting of cytokines, lymphokines, co-stimulatory molecules and nucleic acids encoding them. A pharmaceutical composition comprising the CEA agonist polypeptide according to claim 1 or 2 and a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition comprising a nucleic acid according to any one of claims 3 to 11 and a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition comprising a vector according to any one of claims 12 to 18 and a pharmaceutically acceptable diluent or carrier. 23. A pharmaceutical composition comprising a cell according to any one of claims 19 to 22 and a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition comprising the mixture of any of claims 23 to 26 and a pharmaceutically acceptable diluent or carrier. 32. The pharmaceutical composition according to any one of claims 27 to 31, further comprising an adjuvant. (I) CEA protein or a fragment thereof;
(Ii) the CEA agonist polypeptide according to claim 1 or 2;
(Iii) CEA epitope;
(Iv) a modified CEA epitope;
(V) a cell that expresses a CEA protein or a fragment thereof, a CEA agonist polypeptide, a CEA epitope, or a modified CEA epitope according to claim 1 or 2; and (vi) a CEA protein or a fragment thereof, according to claim 1 or 2. A method of inducing an immune response in an animal against a substance selected from the group consisting of a cell bound with a CEA agonist polypeptide, a CEA epitope, or a modified CEA epitope;
3. A method comprising administering to said animal a CEA agonist polypeptide or fragment thereof according to claim 1 or 2 in an amount sufficient to induce an immune response. (I) CEA protein or a fragment thereof;
(Ii) the CEA agonist polypeptide according to claim 1 or 2;
(Iii) CEA epitope;
(Iv) a CEA protein or a fragment thereof, a cell expressing the CEA agonist polypeptide, CEA epitope or modified CEA epitope of claim 1 or 2; and (v) a CEA protein or a fragment thereof, of claim 1 or 2. A method of inducing an immune response in an animal against a substance selected from the group consisting of cells that bind a CEA agonist polypeptide, a CEA epitope, or a modified CEA epitope;
12. A method comprising administering to said animal a nucleic acid according to any of claims 3 to 11 in an amount sufficient to induce an immune response. (I) CEA protein or a fragment thereof;
(Ii) the CEA agonist polypeptide according to claim 1 or 2;
(Iii) CEA epitope;
(Iv) a modified CEA epitope;
(V) a cell that expresses a CEA protein or a fragment thereof, a CEA agonist polypeptide, a CEA epitope, or a modified CEA epitope according to claim 1 or 2; and (vi) a CEA protein or a fragment thereof, according to claim 1 or 2. A method of inducing an immune response in an animal against a substance selected from the group consisting of a cell bound with a CEA agonist polypeptide, a CEA epitope, or a modified CEA epitope;
19. A method comprising administering to said animal a vector according to any one of claims 12 to 18 in an amount sufficient to induce an immune response. (I) CEA protein or a fragment thereof;
(Ii) the CEA agonist polypeptide according to claim 1 or 2;
(Iii) CEA epitope;
(Iv) a CEA protein or a fragment thereof, a cell expressing the CEA agonist polypeptide, CEA epitope or modified CEA epitope of claim 1 or 2; and (v) a CEA protein or a fragment thereof, of claim 1 or 2. A method of inducing an immune response in an animal against a substance selected from the group consisting of cells that bind a CEA agonist polypeptide, a CEA epitope, or a modified CEA epitope;
23. A method comprising administering to the animal the cells of any one of claims 19 to 22 in an amount sufficient to induce an immune response. (I) CEA protein or a fragment thereof;
(Ii) the CEA agonist polypeptide according to claim 1 or 2;
(Iii) CEA epitope;
(Iv) a modified CEA epitope;
(V) a cell that expresses a CEA protein or a fragment thereof, a CEA agonist polypeptide, a CEA epitope, or a modified CEA epitope according to claim 1 or 2; and (vi) a CEA protein or a fragment thereof, according to claim 1 or 2. A method of inducing an immune response in an animal against a substance selected from the group consisting of a cell bound with a CEA agonist polypeptide, a CEA epitope, or a modified CEA epitope;
27. A method comprising administering to the animal a mixture of any of claims 23 to 26 in an amount sufficient to induce an immune response. (I) CEA protein or a fragment thereof;
(Ii) the CEA agonist polypeptide according to claim 1 or 2;
(Iii) CEA epitope;
(Iv) a CEA protein or a fragment thereof, a cell expressing the CEA agonist polypeptide, CEA epitope or modified CEA epitope of claim 1 or 2; and (v) a CEA protein or a fragment thereof, of claim 1 or 2. A method of inducing an immune response in an animal against a substance selected from the group consisting of cells that bind a CEA agonist polypeptide, a CEA epitope, or a modified CEA epitope;
33. A method comprising administering to the animal a composition according to any one of claims 27 to 32 in an amount sufficient to induce an immune response. A method of inhibiting a CEA epitope-expressing cancer cell in a patient, comprising administering to the patient an effective amount of the CEA agonist polypeptide of claim 1 or 2. 12. A method of inhibiting CEA epitope-expressing cancer cells in a patient, comprising administering to the patient an effective amount of a nucleic acid according to any of claims 3 to 11. 19. A method of inhibiting CEA epitope-expressing cancer cells in a patient, comprising administering to the patient an effective amount of the vector of any one of claims 12-18. 23. A method of inhibiting a CEA epitope-expressing cancer cell in a patient, comprising administering to the patient an effective amount of a cell according to any one of claims 19 to 22. 27. A method of inhibiting CEA epitope-expressing cancer cells in a patient, comprising administering to the patient an effective amount of the mixture of any one of claims 23-26. 33. A method of inhibiting a CEA epitope-expressing cancer cell in a patient, comprising administering to the patient an effective amount of the composition of any one of claims 27-32. The method according to any one of claims 39 to 44, wherein the cancer cells are gastrointestinal cancer cells, breast cancer cells, pancreatic cancer cells, bladder cancer cells, ovarian cancer cells, lung cancer cells, or prostate cancer cells. A method for treating cancer, comprising the method according to any one of claims 39 to 45. Use of a CEA agonist polypeptide according to claim 1 for the manufacture of a medicament for treating cancer. 50. The use according to claim 47, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 1. Use of an isolated, purified or recombinant nucleic acid sequence having the sequence of SEQ ID NO: 2 for the manufacture of a medicament for treating cancer.

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